ReducedShmt2expression impairs mitochondrial folate accumulation and respiration, and leads to uracil accumulation in mouse mitochondrial DNA

Abstract

Background. Adequate cellular thymidylate (dTMP) pools are essential for preservation of nuclear and mitochondrial genome stability. Previous studies have indicated that disruption in dTMP synthesis in the nucleus leads to increased uracil misincorporation into DNA affecting genome stability. To date, the effects of impaired mitochondrial dTMP synthesis in non-transformed tissues have been understudied. Objective. This study aimed to determine the effects of decreased serine hydroxymethyltransferase 2 (Shmt2) expression and dietary folate deficiency on mitochondrial DNA integrity and mitochondrial function in mouse tissues. Methods. Liver mitochondrial DNA (mtDNA) content, and uracil content in liver mtDNA was measured in Shmt2+/- and Shmt2+/+ mice weaned onto either a folate-sufficient control diet (2 mg/kg folic acid, C) or a modified diet lacking folic acid (0 mg/kg folic acid, FD) for 7 wks. Shmt2+/- and Shmt2+/+ mouse embryonic fibroblasts (MEF cells) were cultured in defined culture medium containing either 0 or 25 nM folate to assess proliferative capacity and mitochondrial function. Results. Shmt2+/- mice exhibited 48-67% reduction in SHMT2 protein levels in tissues. Interestingly, Shmt2+/- mice consuming the folate-sufficient C diet exhibited a 25% reduction in total folate in liver mitochondria. There was also a >20-fold increase in uracil in liver mtDNA in Shmt2+/- mice consuming the C diet, and dietary folate deficiency also increased uracil content in mouse liver mtDNA from both Shmt2+/+ and Shmt2+/- mice. Furthermore, decreased Shmt2 expression in MEF cells reduced cell proliferation, mitochondrial membrane potential, and oxygen consumption rate. Conclusions. This study demonstrates that Shmt2 heterozygosity and dietary folate deficiency impair mitochondrial dTMP synthesis, as evidenced by the increased uracil in mtDNA. In addition, Shmt2 heterozygosity impairs mitochondrial function in MEF cells. These findings suggest that elevated uracil in mtDNA may impair mitochondrial function.

Vitamin B12 status and folic acid supplementation influence mitochondrial heteroplasmy levels in mice as they age

“…We found higher levels of uracil in the mtDNA of all mice fed a vitamin B12 deficient diet and in Cd320 -/mice fed the vitamin B12 replete diet. The consequences of uracil incorporation into mtDNA have not been resolved, though uracil in mtDNA is associated with impaired maximal oxidative phosphorylation capacity 66,67 . The mitochondria and the nucleus have independent DNA repair systems.…”

“…Uracil content measures were performed as described 66 . Briefly, 6 primer sets were designed to amplify the whole mitochondrial genome in 6 fragments of differing lengths.…”